Identification of a putative α-galactoside ß-(1 → 3)-galactosyltransferase involved in the biosynthesis of galactomannan side chain of glucuronoxylomannogalactan in Cryptococcus neoformans.

The cell surface of Cryptococcus neoformans is covered by a thick capsular polysaccharide. The capsule is the most important virulence factor of C. neoformans; however, the complete mechanism of its biosynthesis is unknown. The capsule is composed of glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal). As GXM is the most abundant component of the capsule, many studies have focused on GXM biosynthesis. However, although GXMGal has an important role in virulence, studies on its biosynthesis are scarce. Herein, we have identified a GT31 family ß-(1 → 3)-galactosyltransferase Ggt2, which is involved in the biosynthesis of the galactomannan side chain of GXMGal. Comparative analysis of GXMGal produced by a ggt2 disruption strain revealed that Ggt2 is a glycosyltransferase that catalyzes the initial reaction in the synthesis of the galactomannan side chain of GXMGal. The ggt2 disruption strain showed a temperature-sensitive phenotype at 37°C, indicating that the galactomannan side chain of GXMGal is important for high-temperature stress tolerance in C. neoformans. Our findings provide insights into complex capsule biosynthesis in C. neoformans.

Discovery of α-(1→6)-linked mannan structures resembling yeast N-glycan outer chains in Aspergillus fumigatus mycelium.

The cellular surface of the pathogenic filamentous fungus Aspergillus fumigatus is enveloped in a mannose layer, featuring well-established fungal-type galactomannan and O-mannose-type galactomannan. This study reports the discovery of cell wall component in A. fumigatus mycelium, which resembles N-glycan outer chains found in yeast. The glycosyltransferases involved in its biosynthesis in A. fumigatus were identified, with a focus on two key α-(1→2)-mannosyltransferases, Mnn2 and Mnn5, and two α-(1→6)-mannosyltransferases, Mnn9 and Van1. In vitro examination revealed the roles of recombinant Mnn2 and Mnn5 in transferring α-(1→2)-mannosyl residues. Proton nuclear magnetic resonance (1H-NMR) analysis of cell wall extracts from the ∆mnn2∆mnn5 strain indicated the existence of an α-(1→6)-linked mannan backbone in the A. fumigatus mycelium, with Mnn2 and Mnn5 adding α-(1→2)-mannosyl residues to this backbone. The α-(1→6)-linked mannan backbone was absent in strains where mnn9 or van1 was disrupted in the parental ∆mnn2∆mnn5 strain in A. fumigatus. Mnn9 and Van1 functioned as α-(1→6)-linked mannan polymerases in heterodimers when co-expressed in Escherichia coli, indicating their crucial role in biosynthesizing the α-(1→6)-linked mannan backbone. Disruptions of these mannosyltransferases did not affect fungal-type galactomannan biosynthesis. This study provides insights into the complexity of fungal cell wall architecture and a better understanding of mannan biosynthesis in A. fumigatus. IMPORTANCE: This study unravels the complexities of mannan biosynthesis in A. fumigatus, a key area for antifungal drug discovery. It reveals the presence of α-(1→6)-linked mannan structures resembling yeast N-glycan outer chains in A. fumigatus mycelium, offering fresh insights into the fungal cell wall's design. Key enzymes, Mnn2, Mnn5, Mnn9, and Van1, are instrumental in this process, with Mnn2 and Mnn5 adding specific mannose residues and Mnn9 and Van1 assembling the α-(1→6)-linked mannan structures. Although fungal-type galactomannan's presence in the cell wall is known, the existence of an α-(1→6)-linked mannan adds a new dimension to our understanding. This intricate web of mannan biosynthesis opens avenues for further exploration and enhances our understanding of fungal cell wall dynamics, paving the way for targeted drug development.

Complete genome sequence of Bacillus subtilis subsp. natto NARUSE using PacBio sequencing.

We report the complete genome sequence of Bacillus subtilis subsp. natto NARUSE, which has been traditionally employed for fermenting soybeans in Japan. The genome was sequenced using the PacBio system, yielding a sequence, yielding a sequence length of 4,148,793 nucleotides for the circular chromosome and 62,770 nucleotides for the plasmid.

Identification of galactofuranose antigens such as galactomannoproteins and fungal-type galactomannan from the yellow koji fungus (Aspergillus oryzae).

Filamentous fungi belonging to the genus Aspergillus are known to possess galactomannan in their cell walls. Galactomannan is highly antigenic to humans and has been reported to be involved in the pathogenicity of pathogenic filamentous fungi, such as A. fumigatus, and in immune responses. In this study, we aimed to confirm the presence of D-galactofuranose-containing glycans and to clarify the biosynthesis of D-galactofuranose-containing glycans in Aspergillus oryzae, a yellow koji fungus. We found that the galactofuranose antigen is also present in A. oryzae. Deletion of ugmA, which encodes UDP-galactopyranose mutase in A. oryzae, suppressed mycelial elongation, suggesting that D-galactofuranose-containing glycans play an important role in cell wall integrity in A. oryzae. Proton nuclear magnetic resonance spectrometry revealed that the galactofuranose-containing sugar chain was deficient and that core mannan backbone structures were present in ΔugmA A. oryzae, indicating the presence of fungal-type galactomannan in the cell wall fraction of A. oryzae. The findings of this study provide new insights into the cell wall structure of A. oryzae, which is essential for the production of fermented foods in Japan.

Identification of an α-(1→6)-Mannosyltransferase Contributing To Biosynthesis of the Fungal-Type Galactomannan α-Core-Mannan Structure in Aspergillus fumigatus.

Fungal-type galactomannan, a cell wall component of Aspergillus fumigatus, is composed of α-(1→2)-/α-(1→6)-linked mannan and ß-(1→5)-/ß-(1→6)-linked galactofuran side chains. Recently, CmsA and CmsB were identified as the α-(1→2)-mannosyltransferases involved in the biosynthesis of the α-core-mannan. However, the α-(1→6)-mannosyltransferase involved in the biosynthesis of the α-core-mannan has not been identified yet. In this study, we analyzed 9 putative α-(1→6)-mannosyltransferase gene disruption strains of A. fumigatus. The ΔanpA strain resulted in decreased mycelial elongation and reduced conidia formation. Proton nuclear magnetic resonance analysis revealed that the ΔanpA strain failed to produce the α-core-mannan of fungal-type galactomannan. We also found that recombinant AnpA exhibited much stronger α-(1→6)-mannosyltransferase activity toward α-(1→2)-mannobiose than α-(1→6)-mannobiose in vitro. Molecular simulations corroborated the fact that AnpA has a structure that can recognize the donor and acceptor substrates suitable for α-(1→6)-mannoside bond formation and that its catalytic activity would be specific for the elongation of the α-core-mannan structure in vivo. The identified AnpA is similar to Anp1p, which is involved in the elongation of the N-glycan outer chain in budding yeast, but the building sugar chain structure is different. The difference was attributed to the difference in substrate recognition of AnpA, which was clarified by simulations based on protein conformation. Thus, even proteins that seem to be functionally identical due to amino acid sequence similarity may be glycosyltransferase enzymes that make different glycans upon detailed analysis. This study describes an example of such a case. IMPORTANCE Fungal-type galactomannan is a polysaccharide incorporated into the cell wall of filamentous fungi belonging to the subphylum Pezizomycotina. Biosynthetic enzymes of fungal-type galactomannan are potential targets for antifungal drugs and agrochemicals. In this study, we identified an α-(1→6)-mannosyltransferase responsible for the biosynthesis of the α-core-mannan of fungal-type galactomannan, which has not been known for a long time. The findings of this study shed light on processes that shape this cellular structure while identifying a key enzyme essential for the biosynthesis of fungal-type galactomannan.

Mnt1, an α-(1 → 2)-mannosyltransferase responsible for the elongation of N-glycans and O-glycans in Aspergillus fumigatus.

The fungal cell wall is necessary for survival as it serves a barrier for physical protection. Therefore, glycosyltransferases responsible for the synthesis of cell wall polysaccharides may be suitable targets for drug development. Mannose is a monosaccharide that is commonly found in sugar chains in the walls of fungi. Mannose residues are present in fungal-type galactomannan, O-glycans, N-glycans, glycosylphosphatidylinositol anchors, and glycosyl inositol phosphorylceramides in Aspergillus fumigatus. Three genes that are homologous to α-(1 â†’ 2)-mannosyltransferase genes and belong to the glycosyltransferase family 15 were found in the A. fumigatus strain, Af293/A1163, genome: cmsA/ktr4, cmsB/ktr7, and mnt1. It is reported that the mutant ∆mnt1 strain exhibited a wide range of properties that included high temperature and drug sensitivity, reduced conidia formation, leakage at the hyphal tips, and attenuation of virulence. However, it is unclear whether Mnt1 is a bona fide α-(1 â†’ 2)-mannosyltransferase and which mannose residues are synthesized by Mnt1 in vivo. In this study, we elucidated the structure of the Mnt1 reaction product, the structure of O-glycan in the Δmnt1 strain. In addition, the length of N-glycans attached to invertase was evaluated in the Δmnt1 strain. The results indicated that Mnt1 functioned as an α-(1 â†’ 2)-mannosyltransferase involved in the elongation of N-glycans and synthesis of the second mannose residue of O-glycans. The widespread abnormal phenotype caused by the disruption of the mnt1 gene is the combined result of the loss of mannose residues from O-glycans and N-glycans. We also clarified the enzymatic properties and substrate specificity of Mnt1 based on its predicted protein structure.

Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillus fumigatus.

Fungal cell walls and their biosynthetic enzymes are potential targets for novel antifungal agents. Recently, two mannosyltransferases, namely core-mannan synthases A (CmsA/Ktr4) and B (CmsB/Ktr7), were found to play roles in the core-mannan biosynthesis of fungal-type galactomannan. CmsA/Ktr4 is an α-(1→2)-mannosyltransferase responsible for α-(1→2)-mannan biosynthesis in fungal-type galactomannan, which covers the cell surface of Aspergillus fumigatus Strains with disrupted cmsA/ktr4 have been shown to exhibit strongly suppressed hyphal elongation and conidiation alongside reduced virulence in a mouse model of invasive aspergillosis, indicating that CmsA/Ktr4 is a potential novel antifungal candidate. In this study we present the 3D structures of the soluble catalytic domain of CmsA/Ktr4, as determined by X-ray crystallography at a resolution of 1.95 Å, as well as the enzyme and Mn2+/GDP complex to 1.90 Å resolution. The CmsA/Ktr4 protein not only contains a highly conserved binding pocket for the donor substrate, GDP-mannose, but also has a unique broad cleft structure formed by its N- and C-terminal regions and is expected to recognize the acceptor substrate, a mannan chain. Based on these crystal structures, we also present a 3D structural model of the enzyme-substrate complex generated using docking and molecular dynamics simulations with α-Man-(1→6)-α-Man-(1→2)-α-Man-OMe as the model structure for the acceptor substrate. This predicted enzyme-substrate complex structure is also supported by findings from single amino acid substitution CmsA/Ktr4 mutants expressed in ΔcmsA/ktr4 A. fumigatus cells. Taken together, these results provide basic information for developing specific α-mannan biosynthesis inhibitors for use as pharmaceuticals and/or pesticides.

Immunohistochemical Pharmacokinetics of the Anti-diabetes Drug Alogliptin in Rat Kidney and Liver.

Alogliptin is one of a new class of therapeutic agents for type 2 diabetes called dipeptidyl peptidase-4 inhibitors. Here, we used immunohistochemistry to investigate the pharmacokinetics of alogliptin at the cell and tissue levels in the rat kidney and liver. One hour after alogliptin administration, the most noticeable immunoreactivity in the kidney was a moderate-to-strong staining in proximal tubule S3 segment epithelial cells. On the other hand, immunostaining was found only in the microvilli of S1 and S2 segment cells. Immunoreactivity was also observed in the glomerulus and distal tubules. Positive cells and almost negative cells coexisted in the collecting ducts. Twenty-four hours after administration, moderate immunostaining remained in the S3 segment but staining in other regions had almost disappeared. In the liver 1 hr after administration, hepatocyte staining differed in the hepatic lobule, with zone III being stronger than zone I. Immunostaining had almost disappeared 24 hr after administration. These findings suggest that alogliptin reabsorption at the kidney and uptake at the hepatocyte vary from region to region and that one or more types of transporter are involved in these processes. In addition, long-term alogliptin use may cause the drug to accumulate in S3 segment, leading to adverse events.

Unique hexameric structure of copper-containing nitrite reductase of an anammox bacterium KSU-1.

Anaerobic ammonium oxidation (anammox) and denitrification are two different microbial reactions that form nitrogen gas. The initial step in the anammox reaction-reduction of nitrite to nitric oxide-is thought to be catalyzed by homologs of dissimilatory nitrite reductase, which is known to be involved in denitrification. Here, we reveal the crystal structure of the copper-containing nitrite reductase (CuNIR) of strain KSU-1, an anammox bacterium. CuNIR had a unique homohexameric structure with three disulfide bridges between homotrimers, although the trimer was similar to that of known CuNIRs. Kinetic and mutagenesis analyses suggested that the hexameric structure is important for the electron transfer reaction.

Nitric Oxide Production from Nitrite Reduction and Hydroxylamine Oxidation by Copper-containing Dissimilatory Nitrite Reductase (NirK) from the Aerobic Ammonia-oxidizing Archaeon, Nitrososphaera viennensis.

Aerobic ammonia-oxidizing archaea (AOA) play a crucial role in the global nitrogen cycle by oxidizing ammonia to nitrite, and nitric oxide (NO) is a key intermediate in AOA for sustaining aerobic ammonia oxidation activity. We herein heterologously expressed the NO-forming, copper-containing, dissimilatory nitrite reductase (NirK) from Nitrososphaera viennensis and investigated its enzymatic properties. The recombinant protein catalyzed the reduction of 15NO2- to 15NO, the oxidation of hydroxylamine (15NH2OH) to 15NO, and the production of 14-15N2O from 15NH2OH and 14NO2-. To the best of our knowledge, the present study is the first to document the enzymatic properties of AOA NirK.

Anammox Organism KSU-1 Expresses a Novel His/DOPA Ligated Cytochrome c.

Anammox is a bacterial energy metabolic process that forms N2 gas from nitrite and ammonium ions. The enzymatic mechanisms of anammox have been gradually revealed; however, the electron transport chain in anammox bacteria remains poorly understood. In the present study, we purified and characterized two low-molecular-weight c-type cytochromes from an enriched culture of the anammox bacterium strain, KSU-1. Their genes, KSU1_B0428 and KSU1_C0855, were identified in the KSU-1 genome, and their recombinant proteins were characterized. KSU1_B0428 is a typical c-type cytochrome with a His/Met coordinated heme, acting as an electron transfer protein. In contrast, KSU1_C0855 could not be assigned as a known cytochrome and its heme was suggested to have an uncommon axial ligand set. Crystal structural analyses of C0855 clearly showed that its heme iron is coordinated by His15 as a fifth ligand. Moreover, the sixth coordination site is occupied by the aromatic ring of Tyr60, and an unassignable electron density that is inseparable with that of aromatic carbon of Tyr60 was found. The additional electron density was assigned to an O atom by molecular mass analyses. Therefore, Tyr60 would be chemically modified to 3,4-dihydroxyphenylalanine and bound to the Fe atom. We revealed that an anammox bacterium strain KSU-1 expresses a novel cytochrome c having an unprecedented His/3,4-dihydroxyphenylalanine coordinating heme. The expression of the novel c-type cytochrome might be required for the redox reaction of the anammox process.

Impact of aerobic acclimation on the nitrification performance and microbial community of landfill leachate sludge.

Nitrogenous pollution of water is regarded as a global environmental problem, and nitrogen removal has become an important issue in wastewater treatment processes. Landfill leachate is a typical large source of nitrogenous wastewater. Although the characteristics of leachate vary according to the age of the landfill, leachates of mature landfill have high concentrations of nitrogenous compounds. Most nitrogen in these leachates is in the form of ammonium nitrogen. In this study, we investigated the bacterial community of sludge from a landfill leachate lagoon by pyrosequencing of the bacterial 16S rRNA gene. The sludge was acclimated in a laboratory-scale reactor with aeration using a mechanical stirrer to promote nitrification. On 149 days, nitrification was achieved and then the bacterial community was also analyzed. The bacterial community was also analyzed after nitrification was achieved. Pyrosequencing analyses revealed that the abundances of ammonia-oxidizing and nitrite-oxidizing bacteria were increased by acclimation and their total proportions increased to >15% of total biomass. Changes in the sulfate-reducing and sulfur-oxidizing bacteria were also observed during the acclimation process. The aerobic acclimation process enriched a nitrifying microbial community from the landfill leachate sludge. These results suggested that the aerobic acclimation is a processing method for the nitrification ammonium oxidizing throw the enrichment of nitrifiers. Improvement of this acclimation method would allow nitrogen removal from leachate by nitrification and sulfur denitrification.

Physiological characterization of anaerobic ammonium oxidizing bacterium 'Candidatus†Jettenia caeni'.

To date, six candidate genera of anaerobic ammonium-oxidizing (anammox) bacteria have been identified, and numerous studies have been conducted to understand their ecophysiology. In this study, we examined the physiological characteristics of an anammox bacterium in the genus 'Candidatus Jettenia'. Planctomycete KSU-1 was found to be a mesophilic (20-42.5°C) and neutrophilic (pH 6.5-8.5) bacterium with a maximum growth rate of 0.0020 h(-1) . Planctomycete KSU-1 cells showed typical physiological and structural features of anammox bacteria; i.e. (29) N2 gas production by coupling of (15) NH4 (+) and (14) NO2 (-) , accumulation of hydrazine with the consumption of hydroxylamine and the presence of anammoxosome. In addition, the cells were capable of respiratory ammonification with oxidation of acetate. Notably, the cells contained menaquinone-7 as a dominant respiratory quinone. Proteomic analysis was performed to examine underlying core metabolisms, and high expressions of hydrazine synthase, hydrazine dehydrogenase, hydroxylamine dehydrogenase, nitrite/nitrate oxidoreductase and carbon monoxide dehydrogenase/acetyl-CoA synthase were detected. These proteins require iron or copper as a metal cofactor, and both were dominant in planctomycete KSU-1 cells. On the basis of these experimental results, we proposed the name 'Ca. Jettenia caeni' sp. nov. for the bacterial clade of the planctomycete KSU-1.

Reduction of nitric oxide catalyzed by hydroxylamine oxidoreductase from an anammox bacterium.

The hydroxylamine oxidoreductase (HAO) from the anammox bacterium, Candidatus Kuenenia stuttgartiensis has been reported to catalyze the oxidation of hydroxylamine (NH2OH) to nitric oxide (NO) by using bovine cytochrome c as an oxidant. In contrast, we investigated whether the HAO from anammox bacterium strain KSU-1 could catalyze the reduction of NO with reduced benzyl viologen (BVred) and the NO-releasing reagent, NOC 7. The reduction proceeded, resulting in the formation of NH2OH as a product. The oxidation rate of BVred was proportional to the concentration of BVred itself for a short period in each experiment, a situation that was termed quasi-steady state. The analyses of the states at various concentrations of HAO allowed us to determine the rate constant for the catalytic reaction, (2.85 ± 0.19) × 10(5) M(-1) s(-1), governing NO reduction by BVred and HAO, which was comparable to that reported for the HAO from the ammonium oxidizer, Nitrosomonas with reduced methyl viologen. These results suggest that the anammox HAO functions to adjust anammox by inter-conversion of NO and NH2OH depending on the redox potential of the physiological electron transfer protein in anammox bacteria.

Anammox organism KSU-1 expresses a NirK-type copper-containing nitrite reductase instead of a NirS-type with cytochrome cd1.

Anaerobic ammonium oxidation (anammox) and denitrification are two distinct microbial reactions relevant to the global nitrogen cycle. The proposed initial step of the anammox reactions, reduction of nitrite to nitric oxide, has been postulated to be identical to that in denitrification catalyzed by the dissimilatory nitrite reductase of the cytochrome cd(1)-type. Here, we characterized the copper-containing nitrite reductase homolog encoded by nirK detected in the genome of an anammox bacterium strain KSU-1. We hypothesize that this NirK-type nitrite reductase, rather than a nitrite reductase of the cytochrome cd(1)-type (NirS), is likely to catalyze nitrite reduction in anammox organism KSU-1.

High rate nitrogen removal by the CANON process at ambient temperature.

Completely autotrophic nitrogen removal over nitrite (CANON) is a cost-effective nitrogen removal process. Implementation of the CANON process relies on the cooperation of ammonium-oxidizing and Anammox bacteria, as well as the inhibition of nitrite-oxidizing bacteria. Strict limitations on dissolved oxygen (DO) concentration in the reactor, and the addition of sufficient inorganic carbon in the influent, were adopted as the main operational strategies. The reactor was fed with synthetic inorganic wastewater composed mainly of NH(4)(+)-N, and operated for 106 days. Stable nitrogen removal rates (NRR) of around 1.4 kg N m(-3) d(-1) were obtained at ambient temperature. Morphological characteristics and analysis of bacterial community confirmed the formation of functional outer aerobic and inner anaerobic granular sludge, providing evidence of stable nitrogen removal.

Effect of temperature on low-strength wastewater treatment by UASB reactor using poly(vinyl alcohol)-gel carrier.

The feasibility of treating low-strength wastewater with an up-flow anaerobic sludge blanket (UASB) reactor, using a poly(vinyl alcohol)-gel carrier, at various temperatures and hydraulic retention times (HRTs) was examined. The temperature was decreased from 35°C to 25°C and then to 15°C. The HRT was reduced from 2.0 h to 0.22 h. The COD removal rate reached 28 kg-COD m(-3)d(-1) at 35°C, 16 kg-COD m(-3)d(-1) at 25°C, and 6 kg-COD m(-3)d(-1) at 15°C. The COD removal rate was reduced by half for each temperature reduction of 10°C.

Reject water treatment by improvement of whole cell anammox entrapment using polyvinyl alcohol/alginate gel.

Reject water treatment performance was investigated by whole cell anammox sludge entrapped polyvinyl alcohol/sodium alginate gel in the stirred tank reactor (STR). The whole experiment was conducted through Phase 1 and Phase 2 in which synthetic wastewater and modified reject water were used as feeding medium, respectively. The anammox reactor demonstrated quick start-up after 22 days as well as stable and relatively high nitrogen removal rate of more than 8.0 kg-N m(-3) day(-1) during the two both phases even under moderately low temperature of 25 ± 0.5°C during the last 2 months of Phase 2. The matured brownish red PVA beads had good characteristics with buoyant density of 1.10 g cm(-3), settling velocity of 141 m h(-1) and diameter of 4 mm. The bacterial community was identified by 16S rDNA analysis revealing the concurrent existence of KSU-1 and new kind anammox bacterium Kumadai-I after changing influent from synthetic wastewater to reject water. It was speculated that Kumadai-I might play a role as "promotion" factor together with KSU-1 on high nitrogen removal rate. These results demonstrate the potential application of whole cell anammox entrapment by PVA/alginate gel for achieving stable and high-rate nitrogen removal from high ammonium with low C/N ratio contained wastewaters, such as reject water, digester liquor or landfill leachate.

Nitrogen removal performance of a hybrid anammox reactor.

The anaerobic ammonium oxidation (anammox) process has attracted considerable attention in recent years as an alternative to conventional nitrogen removal technologies. In this study, an innovative hybrid reactor combining fluidized and fixed beds for anammox treatment was developed. The fluidized bed was mechanically stirred and the gaseous product could be rapidly released from the anammox sludge to prevent washout of the sludge caused by flotation. The fixed bed comprising a non-woven biomass carrier could efficiently catch sludge to reduce washout. During the operation, nitrogen loading rates to the reactor were increased to 27.3 kg N/m(3)/d, with total nitrogen removal efficiencies of 75%. The biomass concentration in the fluidized bed reached 26-g VSS/L. Anammox granules were observed in the reactors, with settling velocities and sludge volumetric index of 27.3 ± 6.5m/h and 23 mL/g, respectively. Quantification of extracellular polymeric substances revealed the anammox granules contained a significant amount of extracellular proteins.

Factors affecting the treatment of reject water by the anammox process.

Reject water from a municipal wastewater treatment plant was treated using a stirred tank anammox reactor after being treated by a partial nitrification reactor. The results indicated the variations in the influent NO(2)(-)-N to NH(4)(+)-N ratio had a negative effect on reactor performance, especially when the T-N concentrations were high. Influent total organic carbon concentrations greater than 50mg/L were proven to have a serious effect on the nitrogen removal efficiencies of the anammox reactor. Observations by scanning electron microscope showed that the surface of the anammox granular sludge was covered by some materials, possibly the effluent SS contained in the partial nitrified reject water. Furthermore, the study of the bacterial composition of the anammox granular sludge showed that the anammox bacterium, Planctomycete KSU-1, was dominant, even during the inhibition phase.